The present invention relates to isolation, purification and analysis of ribonucleic acid, RNA. In particular, the present invention relates to isolation of RNA from biological sources containing a mixture of biological substances such as proteins, carbohydrates and nucleic acids, and to purification of the isolated RNA.
Ribonucleic acid (RNA), working together with deoxyribonucleic acid (DNA), functions in the control of life processes. In most living organisms, genetic information is stored as DNA, which is a stable, double-stranded polynucleotide. Expression of the genetic information stored in DNA is accomplished by transcription of RNA from a DNA template, followed by translation of the RNA-encoded information into protein.
Gene expression is regulated at both the transcriptional and the translational level. The quantity and characteristics of RNA in a cell is, therefore, an extremely important indicator of gene regulatory events taking place in that cell. As a unit of gene expression, the quantity of a given RNA sequence in cells indicates the level of expression of a given DNA sequence. A great deal of information regarding the physiological state of cells comprising organisms can be gained by determining the presence, quantity, nucleotide sequence and/or structure of a specific RNA. Therefore, purification and analysis of RNA is critical to the study and elucidation of mechanisms by which gene expression is regulated.
Because of the structural similarity between DNA and RNA, previous RNA purification methods have often comprised isolating DNA and RNA together from biological sources. One commonly used method for isolating nucleic acids from cells and tissues was the "Sevag" procedure. This method comprises contacting a cell or tissue homogenate with phenol or a mixture of phenol and chloroform, thereby denaturing proteins and precipitating them while leaving nucleic acids in solution. This method, while still widely used, is hazardous, laborious and of limited utility for isolation of RNA from biological sources containing high amounts of ribonuclease (RNAse), an extremely stable enzyme that degrades RNA.
An improved method for isolating intact RNA from ribonuclease-rich tissues was disclosed by Chirgwin et al., Biochemistry, 18: 5924-29 (1979). This method comprises exposing tissue homogenates to concentrated guanidinium thiocyanate and 2-mercaptoethanol, thereby eliminating nucleolytic degradation of RNA by denaturing all of the cellular proteins, including ribonuclease, at a rate which exceeded the rate of RNA hydrolysis by ribonuclease. Although RNA isolated in this manner was biologically active, it was by no means free of contamination by DNA, protein and other cellular materials. Subsequent, often extensive, manipulation was required to further purify the RNA from other cellular contaminants.
A simple method for purifying DNA from certain sources was disclosed by Vogelstein and Gillespie, Proc. Natl. Acad. Sci (USA), 76: 615-19 (1979). DNA-containing sources were incubated with powdered glass in the presence of a strong solution of the chaotropic salt, sodium iodide (NaI), thereby forming a DNA-glass complex. This technique was adapted for purification of DNA from virtually any source by first treating the source with a solution containing a different chaotropic salt, guanidine thiocyanate (GuSCN).
A similar glass-binding technique was disclosed for co-purification of DNA and RNA from certain biological sources. Boom et al., J. Clin. Micro., 28:495-503 (1990). Cells contained within biological sources, such as serum or urine, were lysed by exposure to strong (greater than 5M) solutions of GuSCN in Tris HCl (pH 8.0), containing 0.2 M EDTA and 2.6% (w/v) Triton X-100. DNA and RNA were copurified from the mixture of biological materials by incubation with diatomaceous earth or silica particles, which formed reversible complexes with the DNA and RNA. While this method was of general utility in isolating nucleic acids from biological sources, it did not provide a means for selectively separating RNA from mixtures containing DNA and RNA together and purifying the RNA thus separated.
From the foregoing, it will be appreciated that a simple, effective means is needed for isolating RNA selectively from biological sources in order to avoid contamination with DNA, which can hinder the purification and subsequent analysis of RNA.